Charged particle microscopy mems sample support

ABSTRACT

The present disclosure relates to a sample support device for charged particle microscopy and related methods. The device comprises a substrate and a heating and/or biasing element integrated in or on the substrate to heat (or apply a bias voltage to) a sample when positioned in an observation region of the device. The device comprises a membrane covering an opening in the heater element and/or substrate in the observation region of the device. The membrane is perforated to form at least one hole covered by a graphene layer to form a sample support to place a sample of interest thereon for study. A cap covers the membrane such that a chamber is formed in which the sample can be isolated in a controllable gaseous environment.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the National Phase entry of InternationalPatent Application No. PCT/EP2021/085112 filed Dec. 9, 2021, whichclaims priority to European Patent Application No. 20213255.1 filed Dec.10, 2020, the entire contents of which are hereby incorporated byreference into this application.

TECHNICAL FIELD

The present disclosure relates to the field of charged particlemicroscopy, e.g. transmission electron microscopy, and advanced materialengineering, and, more specifically, the present disclosure relates to acharged particle microscopy sample support with an integrated heaterand/or biasing electrode(s) and a related manufacturing method.

BACKGROUND

Materials and specimens of interest can be studied by charged particlemicroscopy, such as transmission electron microscopy (TEM) or focusedion beam microscopy. For example, TEM can achieve exceptionally highmagnification even with atomic resolution. This enables importantinsights to be gained about the studied specimen, such as characterizingits crystalline structure and orientation, elemental composition andmicrostructure, and various further physical properties of interest.

In TEM, an electron beam is focused onto the specimen, and thetransmitted beam is measured and quantified to construct an image of thestructure of the specimen or to gain elemental and chemical information.However, to hold the specimen in its position, it typically needs to besupported by a sample support grid, of which the signal can interferewith the signal coming from the sample.

The field of transmission electron microscopy (TEM) has significantlyevolved in recent years. For example, aberration correctors haveimproved the attainable resolution, and stability and sensitivity havebeen drastically improved by novel stages and detector designs. However,significant room for improvement remains in the development of sampleholders and supports. The sample support is, ideally, as thin and cleanas possible, so the interference with the sample signal obtained fromthe object under study is limited and known. A sample support gridconventionally comprises a flat surface of a continuous layer, a layerwith holes or a mesh. Depending on the sample type, the sample isdeposited on the support by a drop-casting procedure, or thin sectionsof the sample are deposited on the grid via different sample preparationmethods. Depending on the nature of the sample, the sample is analyzedon the edge of a hole or on top of a support layer, which allowselectrons to pass through.

Conventionally, amorphous carbon-based supports are used, which maytypically have a thickness of about 20 nm. However, such sample supportscan cause a significant and undesirable background signal, which may beparticularly problematic, e.g. for particular types of sample and/orparticularly thin samples. A poor manufacturing reproducibility of thesesupports can furthermore easily lead to failed experiments, or at leasta waste of time, resources and/or samples, which might be avoidable ifonly higher quality supports would be readily available.

Of particular interest are systems that integrate microelectromechanicalsystems (MEMS), for example for heating, biasing and/or solid-gas (orsolid-liquid) interaction experiments. For example, the sample supportmay be specifically adapted to form a miniature laboratory. Amicroheater in close proximity to the specimen under study can heat thesample, for example in a precisely controlled manner. For example,illustrative MEMS-based microheaters, as known in the art, are describedin detail in the U.S. patent application US 2017/062177 A1, and theinternational patent applications WO 2008/141147 A1 and WO 2020/208433A1.

Furthermore, a closed reaction chamber can be formed around the specimenthrough which a gas (or liquid) can flow to study interactions whileimaging the sample. The chamber may, for example, be formed by enclosingthe sample between two chips (at least one of which having a suitablecavity formed therein) to isolate it from the external environment.Examples of such cells, e.g. designed to facilitate the study of liquidsamples, can be found in the patent applications US 2016/042912 A1, US2015/118126 A1 and US 2020/240933 A1, as well as the aforementionedpatent applications WO 2008/141147 A1 and US 2017/062177 A1.

However, this clearly exacerbates the mentioned problems of conventionalsupports, since the electron beam in this case has to pass through twosurfaces, both of which need to meet high standards so as not tointerfere with the obtainable signal quality. For example, in Altantziset al, “Three-Dimensional Quantification of the Facet Evolution of PtNanoparticles in a Variable Gaseous Environment,” Nano Letters 2019, 19,pp. 477-481, such drastic decrease in imaging quality, when a gas cellis used, is clearly presented (cf. e.g. FIG. 1 in said publication).

Furthermore, the material used to form this chamber is typically siliconnitride (Si₃N₄), which has useful properties, e.g. the ability of a thinmembrane of this material to withstand an elevated pressure and hightemperature, its use as an electrical insulator and passivation materialto protect an integrated heater and its ease of manufacturing in aconventional semiconductor process flow. Thus, MEMS sample holderstypically comprise such silicon nitride membranes as electrontransparent windows, e.g. on which the sample is deposited. Even if aclosed chamber is not required, to integrate a MEMS heater in closeproximity to the sample support, this ubiquitous material may still beoften used to form a sample support membrane, e.g. in view of its easeof application.

However, these silicon nitride membranes are typically even thicker thanconventional carbon supports, and its thermal conductivity andelectrical conductivity may be less desirable for this type ofapplication. For example, charging effects due to the poor electricalconductivity of the silicon nitride windows may hamper the acquisitionof high-quality signals in the TEM. Additional distortions in the imagescan be caused by the thermal expansion of the windows and by so-calledbulging effects, in which the height of the window changes due toheating and pressure, causing a loss of focus in the images andenlarging the thickness of the reaction chamber.

For example, EP 2919255 A1 discloses a MEMS-type sample support forconfining gases and/or liquids in a sample holder and/or controlling thetemperature of the specimen while electron imaging. A membrane region ofthe device may consist of one or more thin films, which create aphysical barrier between the environment in the reaction chamber and theenvironment within the electron microscope and/or as a support uponwhich the specimen can be placed.

SUMMARY

It is an object of embodiments of the present disclosure to provide ingood, efficient and/or cheap methods of manufacturing a charged particlebeam microscopy sample support comprising an integrated MEMS structure(i.e. an integrated heater and/or biasing electrode) and the samplesupport obtainable thereby.

It is an aspect of embodiments of the present disclosure that a good,reliable, clean, uniform, and/or homogeneous graphene (mono)layer can beprovided onto a charged particle microscopy sample support with anintegrated heater and/or electrodes to be used to support and/or enclosethe specimen under study.

It is an aspect of embodiments of the present disclosure that a good(e.g. uniform; continuous; large area) coverage and/or high flatness ofthe graphene layer can be achieved on such charged particle microscopysample support.

It is an aspect of embodiments of the present disclosure that thegraphene can be transferred to the charged particle microscopy samplesupport such as to remain intact or substantially intact.

It is an aspect of embodiments of the present disclosure that thegraphene can be transferred to the charged particle microscopy samplesupport without or with little contamination, such as contamination bypolymers or other organic compounds used in the preparation process.

It is an aspect of embodiments of the present disclosure that thegraphene layer to be used to support the sample in the charged particlemicroscopy sample support device can have an low thickness, high thermalconductivity, high electrical conductivity and/or reproducible (good)uniformity.

It is an aspect of embodiments of the present disclosure that thegraphene layer allows a good conduction of heat provided by theintegrated heater to the sample, in use of a device in accordance withembodiments.

It is an aspect of embodiments of the present disclosure that abackground signal obtained when imaging a specimen with TEM or relatedtechniques can be reduced and/or homogenized, thus allowing a simplenumerical subtraction.

It is an aspect of embodiments of the present disclosure that artefactsduring charged particle (e.g. electron) microscopy to a non-conductivespecimen can be reduced or avoided. This may, for example, enable orimprove the study of specimens that are extremely beam sensitive, e.g.including (but not limited to) soft-hard interfaces and/or comprisingdamage-sensitive (in)organic compounds, e.g. capping ligands.

It is an aspect of embodiments of the present disclosure that propertiesof a conventional sample support membrane, e.g. in terms of structuraland temperature stability, such as a silicon nitride membrane, can becombined with the properties of a graphene monolayer as sample support.

A charged particle microscopy sample support device comprising anintegrated heater and/or biasing electrode(s) and a method formanufacturing such device in accordance with embodiments of the presentdisclosure achieve the above objective.

In a first aspect, the present disclosure relates to a sample supportdevice for charged particle microscopy e.g. electron microscopy, e.g.scanning electron microscopy, transmission electron microscopy and/orother electron microscopy modalities. The device comprises a substrateand a heater element, and/or a biasing electrode(s), integrated in or onthe substrate to heat (resp. and/or to apply a bias voltage to) a sampleof interest when positioned in an observation region of the device. Thedevice comprises a membrane covering an opening in the heater elementand/or substrate in the observation region of the device. The membraneis perforated to form at least one hole therein (through the membrane).The device further comprises a graphene layer covering the at least onehole in the membrane to form a sample support onto which the sample canbe placed for study. The sample support device comprises a cap coveringat least the membrane such that a chamber is formed between the cap andthe membrane (and the graphene) in which the sample can be isolated in acontrollable (i.e. controlled in use of the device) gaseous environment.The at least one hole (e.g. each hole) in the membrane is furthermore atleast five times smaller in area than the opening covered by themembrane, which forms the observation region.

The cap may comprise a second substrate to cover at least the membrane,e.g. using a further chip (e.g. in a flipped arrangement or not), e.g.with a suitable window region, so as to complete the cavity enclosure.

The cap may also (additionally or alternatively) comprise a membrane toform a wall of cavity. Such membrane may comprise, or may even consistof, graphene, e.g. may have holes covered by graphene in a mannersimilar to the membrane with graphene-covered holes discussedhereinabove (but e.g. forming a different part of the cavity wall, e.g;such that beam entry and exit windows may be formed by the respectivegraphene covered holes).

In a sample support device in accordance with embodiments of the presentdisclosure, the graphene layer may have a thickness of less than 2 nm.

In a sample support device in accordance with embodiments of the presentdisclosure, the membrane may have a thickness in the range of 5 nm to 90nm.

In a sample support device in accordance with embodiments of the presentdisclosure, the graphene layer may comprise or consist of a number n ofstacked graphene layers, e.g. separately deposited on top of each other,e.g. n mono-atomic layers, in which n is in the range of 2 to 5, e.g. 2or 3.

In a sample support device in accordance with embodiments of the presentdisclosure, the graphene layer may consist of a single graphene layer,e.g. a single mono-atomic layer.

Particularly, the at least one hole in the membrane of the samplesupport device in accordance with embodiments of the present disclosuremay be covered by only the single (monoatomic) graphene layer (or may beonly covered by the single monoatomic graphene layer). However, in asample support device in accordance with other embodiments of thepresent disclosure, the at least one hole in the membrane of the samplesupport device may be covered by more than one (mono-atomic) graphenelayer (e.g. in a stack), but not by more than 5 graphene layers.Nevertheless, embodiments of the present disclosure are not necessarilylimited to either option (i.e. to n=1 or to 1<n<5).

In a sample support device in accordance with embodiments of the presentdisclosure, the membrane may comprise or consists of an amorphoussilicon nitride layer.

In a sample support device in accordance with embodiments of the presentdisclosure, the at least one hole in the membrane may be at least fivetimes smaller in diameter than the opening (forming the observationregion), covered by the membrane.

In a sample support device in accordance with embodiments of the presentdisclosure, the at least one hole in the membrane may be at least tentimes smaller in diameter than the opening covered by the membrane,which forms the observation region.

In a sample support device in accordance with embodiments of the presentdisclosure, the at least one hole in the membrane may be at least tentimes smaller in area than the opening covered by the membrane, whichforms the observation region.

In a sample support device in accordance with embodiments of the presentdisclosure, the at least one hole may have a diameter in the range of 50nm to 5 μm (without necessarily being limited thereto).

In a sample support device in accordance with embodiments of the presentdisclosure, the heater element may comprise a spiral-shaped and/ormeandering electrical conductor.

A sample support device in accordance with embodiments of the presentdisclosure may comprise at least one heat sink element to improve thetemperature stability and/or heating uniformity of a sample when placedon the graphene layer and heated by the heater element.

A sample support device in accordance with embodiments of the presentdisclosure may comprise at least one channel to provide a flow of a gasor a fluid of interest through said chamber.

In a second aspect, the present disclosure relates to a method ofmanufacturing a sample support device for charged particle microscopy.The method comprises providing a substrate having a heater elementand/or a biasing electrode(s) integrated therein or thereon to heat(and/or to apply a bias voltage to) a sample when positioned in anobservation region of the device, and comprising a membrane covering anopening in the heater element and/or substrate in the observationregion. The method comprises perforating the membrane to form at leastone hole through the membrane, e.g. using a focused ion beam (FIB)process step, and transferring a graphene layer onto the membrane tocover the at least one hole in the membrane such that the graphene layerforms a sample support onto which a sample of interest can be placed forstudy, in use of the manufactured device. The method also comprisescovering at least the membrane by a cap such that a chamber is formedbetween the cap and the membrane wherein the sample can be isolated in acontrollable gaseous environment, The at least one hole in the membraneis furthermore at least five times smaller in area than said openingcovered by said membrane (than the opening forming the observationregion).

In a method in accordance with embodiments of the present disclosure,transferring the graphene layer onto the membrane may comprise obtaininga metal foil (or, generally, a suitable carrier material) onto which thegraphene layer is provided (e.g. onto which it may have been grown) andusing a graphene transfer method (e.g. as known in the art) to transferthe graphene layer from the metal foil (carrier material) onto themembrane so as to cover the hole(s), e.g. using a wet transfer method ora dry transfer method. The transfer method may be a polymer-freetransfer method, or may, alternatively, be a transfer method using apolymer support for transfer (e.g. a sacrificial or temporaryintermediate carrier).

In a method in accordance with embodiments of the present disclosure,transferring the graphene layer onto the membrane may comprise:

-   -   obtaining a metal foil onto which the graphene layer is        provided,    -   stabilizing the graphene layer by applying a layer of a        cellulose-based polymer onto the graphene layer,    -   placing the metal foil having respectively the graphene layer        and the cellulose-based polymer layer stacked thereon in or on        an etching solution to dissolve the metal foil supporting the        graphene layer,    -   diluting and/or neutralizing the etching solution after the        metal foil has been dissolved,    -   depositing the graphene layer directly onto the membrane by        placing the substrate underneath the graphene layer floating in        or on the diluted and/or neutralized etching solution and        removing the diluted and/or neutralized etching solution until        the graphene layer settles onto the membrane to cover said at        least one hole.

In a method in accordance with embodiments of the present disclosure,transferring the graphene layer onto the membrane may also comprise drycleaning the substrate, with the graphene layer deposited thereon, inorder to remove residues and/or contaminants (e.g. the cellulose-basedpolymer layer and/or other contaminants).

The dry cleaning may comprise bringing the substrate, with the graphenelayer on the membrane, into direct (physical) contact with activatedcarbon (e.g. embedding the substrate with the graphene layer thereon inactivated carbon) and then heating the activated carbon.

In a method in accordance with embodiments of the present disclosure,transferring the graphene layer onto the membrane may also compriseapplying an annealing treatment, e.g. in high vacuum.

A method in accordance with embodiments of the present disclosure maycomprise covering the electrodes and/or contact pads with a protectivetape or foil, e.g. a polyimide (Kapton) foil, to protect them from theetching solution.

In a method in accordance with embodiments of the present disclosure,transferring the graphene layer onto the membrane, e.g. using thecellulose-based polymer transfer method referred to hereinabove, maycomprise coating the metal foil having the graphene layer attachedthereto with a solution of the cellulose-based polymer using adip-coating or spin-coating method. However, alternatively, anothertransfer method, such as a polymer-free transfer method, may be used.

For example, any cellulose-based polymer that inadvertently isdip-coated directly onto the metal foil on the side opposite of the sidewhere the graphene layer is provided, can be subsequently removed, suchthat the metal foil is exposed and the graphene layer remains covered bythe cellulose-based polymer. In some embodiments, such removal step canbe avoided when using a spin-coating approach.

In a method in accordance with embodiments of the present disclosure,placing the metal foil having respectively the graphene layer and thecellulose-based polymer layer stacked thereon in or on the etchingsolution may comprise placing the metal foil in or on the etchingsolution with the metal foil directed downward, such that the graphenelayer can settle onto the membrane without inversion in the step ofdepositing the graphene layer.

A method in accordance with embodiments of the present disclosure maycomprise drying the substrate with the graphene layer deposited on themembrane before the step of dry cleaning.

In a method in accordance with embodiments of the present disclosure,the activated carbon, in contact with (e.g. embedded in) the substrate,may be heated to a temperature at least 5° C. higher than the meltingtemperature of the cellulose-based polymer and said temperature may bemaintained for at least 30 minutes, e.g. at least 1 hour, e.g. at least2 hours, e.g. at least 4 hours.

For example, the device being manufactured, while in direct contact withthe activated carbon, may be heated to a suitable temperature (e.g. atleast 215° C.) in a vacuum environment, e.g. in a vacuum annealingprocess. For example, the time for such vacuum annealing treatment maybe relatively low, e.g. less than 1 hour may suffice (e.g. 0.5 hour ormore), to achieve good results.

For example, the activated carbon may be provided in the form of a film(or tape, sheet, . . . ) and brought into direct contact with thegraphene layer. Alternatively, the activated carbon may be provided inthe form of a powder (granulate, any other suitable form, . . . ) andthe substrate (with graphene thereon) may be embedded in the activatedcarbon, e.g. which may form a heap or may fill a recipient up to atleast a reasonable height, such that the sample support device can be,entirely, covered by the activated carbon.

The independent and dependent claims describe specific and features ofthe disclosure. Features of the dependent claims can be combined withfeatures of the independent claims and with features of other dependentclaims as deemed appropriate, and not necessarily only as explicitlystated in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative device in accordance with embodiments ofthe present disclosure.

FIG. 2 shows a transverse cross section of an illustrative device inaccordance with embodiments of the present disclosure.

FIG. 3 shows further illustrative devices in accordance with embodimentsof the present disclosure.

FIG. 4 shows a yet further illustrative device in accordance withembodiments of the present disclosure.

FIG. 5 shows schematically a transverse cross section of an illustrativedevice in accordance with embodiments of the present disclosure thatcomprises a closed sample chamber.

FIG. 6 shows schematically a transverse cross section of anotherillustrative device in accordance with embodiments of the presentdisclosure that comprises a closed sample chamber.

FIG. 7 illustrates a method in accordance with embodiments of thepresent disclosure.

FIG. 8 shows steps of a graphene transfer method that can be applied ina method in accordance with embodiments of the present disclosure.

FIG. 9 illustrates an illustrative approach of placing a graphene layeronto a partially finished device under construction in a manufacturingmethod in accordance with embodiments of the present disclosure.

FIG. 10 shows a transmission electron microscopy (TEM) image of a doublegraphene layer covering a membrane hole to form a sample support, in adevice in accordance with embodiments of the present disclosure.

FIG. 11 shows a highly defocused TEM image, corresponding to thegraphene sample support shown in FIG. 10 , in which diffraction featuresare indicative of the presence of a double layer of graphene.

FIG. 12 shows three illustrative TEM images of a sample support, in adevice in accordance with embodiments of the present disclosure, toillustrative robustness and integrity up to relatively hightemperatures.

The drawings are schematic and not limiting. Elements in the drawingsare not necessarily represented on scale. The present disclosure is notnecessarily limited to the specific embodiments of the presentdisclosure as shown in the drawings.

DETAILED DESCRIPTION OF EMBODIMENTS

Notwithstanding the exemplary embodiments described hereinbelow, is thepresent disclosure only limited by the attached claims. The attachedclaims are hereby explicitly incorporated in this detailed description,in which each claim, and each combination of claims as allowed for bythe dependency structure defined by the claims, forms a separateembodiment of the present disclosure.

The word “comprise,” as used in the claims, is not limited to thefeatures, elements or steps as described thereafter, and does notexclude additional features, elements or steps. This therefore specifiesthe presence of the mentioned features without excluding a furtherpresence or addition of one or more features.

In this detailed description, various specific details are presented.Embodiments of the present disclosure can be carried out without thesespecific details. Furthermore, well-known features, elements and/orsteps are not necessarily described in detail for the sake of clarityand conciseness of the present disclosure.

In a first aspect, the present disclosure relates to a charged particlemicroscopy sample support device comprising an integrated heater and/orbiasing electrode. For example, such sample support device may be usedto hold a sample, e.g. a specimen of interest, while being imaged usinga charged particle beam, e.g. an electron beam. The sample supportdevice may be a transmission electron microscopy sample support device.

Referring to FIG. 1 , FIG. 2 and FIG. 3 , an illustrative device 100 inaccordance with embodiments of the present disclosure is shown. Thedevice 100 comprises a MEMS structure, i.e. an integrated heater element101 and/or biasing electrode(s) 1011. In another example, e.g. as shownin FIG. 3 , electrodes 1011 may be arranged on (e.g.) opposite sides ofan observation region 102, such that a voltage over a sample region inbetween the electrodes can be controlled via those electrodes 1011. Thenumber of electrodes 1011 may vary, e.g. embodiments may relate to adevice comprising three electrodes (e.g. a working, counter andreference electrode), a pair of electrodes, or another variation (ofnumber of electrodes and/or design) as known in the art.

The device 100 may comprise a heater element 101 for heating the samplewhen positioned in an observation region 102 on the device. The heaterelement may be adapted to supply heat to the sample by heat conductionand/or ohmic heating. For example, the heater element may comprise anelongate heat and/or electrical conductor, e.g. a metal conductor,between at least two contacts 114, to which heat and/or an electricalcurrent can be applied to heat the sample directly (by heat conduction)or indirectly (by ohmic heating). The (e.g. metal) heater element maycomprise a meandering electrical conductor for conducting an electricalcurrent between the at least two contacts to heat the sample. The heaterelement may have a spiral shape. For example, the metal heater elementmay be a spiral-shaped microheater, e.g. a double spiral shape. Afour-point probe design may be used to allow the temperature to beaccurately measured while simultaneously supplying heat via a current.

The heater element may comprise (for example) two counterrotating spiralarms that continuously (e.g. smoothly) transition in a central region,e.g. showing little or no definition or delineation of the central area,but this central region may also be more clearly delineated, e.g. by abroadening of the conductor in the center, e.g. to form a platform-likestructure (as generally known in the art).

The observation region(s) is/are not necessarily limited to a centralregion of the heater, e.g. may be dispersed over the heater (e.g. alongits unfolded length) and not necessarily limited to the center or acentral part thereof. As shown in FIG. 1 , the observation region(s)1022 may be formed directly in (a hole of opening formed in) a principalconductor forming the heater, but the observation region(s) 1021 mayalso be interspersed in between parts of the conductor (e.g. betweenfolds and/or arms of a meandering and/or spiraling conductor), e.g.formed in an (e.g. insulating) material spacing away different segmentsof the (e.g. same) conductor. A single device 100 in accordance withembodiments may comprise either one type of observation region 1021,1022, or a combination of both.

It is also noted that the heater is not necessarily shaped as a spiralor spiral-like structure (even though this may be desirable to achieve auniform and fast heating). For example, FIG. 4 shows a heater 101 inwhich a current can be supplied via contacts 114 to run over theobservation region 102 to supply heat. If the membrane in theobservation region 102 is sufficiently electrically conductive, acurrent may flow between the heater parts 101. Alternatively, the deviceschematically depicted in FIG. 4 may also be interpreted as showing apair of biasing electrodes 101 on either side of an electricallyinsulating membrane.

In the observation region 102, at least one transparent window (e.g.opening or hole) is formed in the heater element 101, in which thiswindow is covered (or formed) by a membrane 103, such as a (e.g.amorphous) silicon nitride layer or a membrane formed from analternative material, e.g. silicon carbine, boron nitride, carbon,aluminium nitride, silicon dioxide, silicon or any combination thereof.The transparent window is not necessarily provided directly in (i.e.through) the heater element, but may also be disposed in close proximitythereto. For example, FIG. 1 illustrates both windows directly in ametal conductor forming the heater 101 and in an insulator materialspaced in between the conductive track(s) of the heater.

The membrane may also comprise a stack of different layers, e.g. tooptimize its tensile properties. The membrane layer, some layers or alllayers may also be doped to obtain suitable characteristics, as is knownin the art. Thus, different layers may differ in their materialcomposition, but also (or alternatively) in the type of doping and/ordoping levels. To improve the mechanical properties of the membrane, itmay be textured or structured, e.g. by locally varying its thicknessand/or by forming a pattern of structural support elements, e.g. a grid,in, under or on the membrane. Such structural support elements may beformed by locally thicker parts of the membrane and/or by additionalmaterials formed in the membrane stack, e.g. a thin metal layer.

For example, the observation region 102 may comprise a hole or openingin the heater element (and/or its supporting substrate), such that themembrane 103 extends over (covers) the hole in this observation region.This membrane acts as a sample support, albeit indirectly (the samplecan be placed on a graphene layer that is itself supported by themembrane), as well as forms a heatable region, the temperature of whichcan be (precisely) controlled by the heater element. The membrane allowsthe sample to be placed in close proximity to this heat source, whileenabling a substantially unimpeded transmission of a charged particle(e.g. electron) beam used for imaging the sample (e.g. generallytransmitted in a direction perpendicular to the layered structures ofthe device).

The membrane 103 is also perforated in the observation region 102 todefine a hole or holes 105 (i.e. through-hole/s) therein, which arecovered by a graphene layer 104, e.g. monolayer of graphene. Forexample, the hole(s) in the membrane may be at least five, e.g. at leastten, times smaller in area, or in diameter, than the hole (in the heaterand/or substrate) covered by membrane itself. Note that, for the sake ofclarity, the features in the drawings are not necessarily drawn tocorrespond to actual scale and proportions.

The graphene layer is not necessarily limited to a monolayer ofgraphene. For example, the graphene layer (or: layer stack) may compriseor consist of a number n of stacked graphene layers, e.g. n mono-atomiclayers, in which n is in the range of 2 to 5, e.g. 2 or 3. For example,Hydrogen can easily pass through a single monoatomic layer of graphene,due to the size of the hexagonal structure of the graphene. Therefore,stacking at least two graphene layers on top of each other may reduceand/or avoid gas leakage (e.g. of Hydrogen and potentially also otherspecies, e.g. potentially under substantial pressure of a containmentenvironment relative to external pressure).

However, embodiments are not necessarily limited to such multi-layeredgraphene, e.g. only a single graphene monolayer may be usedalternatively, and/or the number of graphene layers may vary (e.g. notnecessarily limited to a maximum of 5 as presented hereinabove).

Even though reference is made to the graphene layer 104 “covering” thehole or holes 105, it will be understood that the hole may be covered oneither side of the opening, e.g. from a top-side or bottom-side (notstrictly excluding a combination thereof either, even though onlycovering a single side of the hole might be more desirable). Forexample, in the examples shown in FIG. 3 , the graphene is applied tocover the hole or holes from a backside of the substrate (e.g. the sidefrom which the substrate has been etched in these examples to form acavity in the substrate in the observation region 102). Even though thisis shown in combination with a pair of biasing electrodes 1011, it willbe understood that embodiments comprising a heater element 101 mayequally have the graphene layer 104 applied on the backside (e.g. thesubstrate-side), or on the frontside (cf. e.g. FIG. 2 ).

For example, the heater element 101 (and/or biasing electrode 1011) maycomprise a metal structure formed on a semiconductor, e.g. silicon,substrate 110. The heat element 101 may be formed by depositing asuitable metal layer, such as molybdenum, platinum, gold, copper, etc.(or alloys thereof), (directly or indirectly) onto the substrate 110,and etching this metal layer to define the structure of the heater. Theheat element may be covered by an insulating layer 111, or encapsulatedbetween insulating layers 111, 112, of a passivating material, such assilicon nitride, for example such as to form a stack of respectively thesubstrate 110, the first insulating layer 112, the metal layer 101 andthe second insulating layer 111 is formed.

Thus, as generally known in the art, the sample support device maycomprise a semiconductor substrate 110, e.g. a (part of a) siliconwafer, e.g. a 400 μm thick single side polished silicon wafer. Onto thesubstrate 110, a first insulator layer 112 may be deposited, e.g. a 200nm thick layer of silicon nitride. Onto the first insulator layer 112, ametal layer may be deposited and structured to form the heater element101, e.g. using a photolithography and reactive ion etching. A secondinsulator layer 111 may be provided on the heater element 101 forinsulation and protection. However, the first insulator layer may beconsidered to be optional.

In the observation region 102, the second insulator layer 111 may be(optionally) thinned to reduce the thickness of the insulator layer inthe observation region. The second insulator layer is not necessarilydeposited in a single step. For example, the patterned metal layer maybe (entirely) covered by the insulating material, after which one ormore windows are etched away (e.g. using again photolithography andRIE), and a thinner layer of the insulating material (the same or adifferent material) may be provided to cover the window(s) with onlythis thin layer. Thus, the heater may be insulated by a relatively thicklayer (e.g. 400 nm), while the observation window is only covered by arelatively thin layer (e.g. 20 nm). The membrane 103 that covers thewindow in the heater 101 may thus be formed directly from the (e.g.second) insulator layer 111 covering the heater or may be deposited in afurther step as a separate layer after (locally) etching away theaforementioned (second) insulator layer 111. It is to be noted thatanalogously the membrane may be formed by (e.g. in) a layer underneaththe heater. Analogously, referring to FIG. 3 , a (set of) biasingelectrode(s) may be deposited on top of an insulator layer from whichthe membrane may be formed. Nonetheless, this is only one possibleexample, and alternative construction methods/designs known in the artfor similar MEMS sample support devices may also be applied toembodiments of the present disclosure.

The substrate and metal layer may be patterned from the backside todefine a cavity underneath the window(s), e.g. such that the membrane103 remains suspended over the cavity, i.e. such that the transparentwindow(s) in the observation region 102 is formed. A part of thesubstrate that is not removed may act as a supporting frame to providestructural integrity to the device, e.g. to support the membrane 103extending over the observation region. For example, the membrane 103 mayhave a tensile stress profile to keep the membrane taut over the cavityin the substrate in the observation region. The frame may, for example,be formed (at least in part) from the substrate, e.g. by a thick regiongenerally around the perimeter of the device, to provide mechanicalsupport to the device. Thus, the device can be handled without damagingthe membrane, and a good contact between the device and a holder can beachieved to use the device in a microscopy system. In some embodiments,some thermal isolation of the membrane may be provided (and/or betweenmembranes when multiple observation regions are provided).

In the observation region, an opening in the metal heater element 101 isthus covered by the membrane 103, e.g. a silicon nitride Si₃N₄ membrane,which is perforated to form a hole(s) 105 therein. This hole or holesmay have a diameter in the range of 50 nm to 5 μm, in the range of 75 nmto 1 μm, e.g. in the range of 100 nm to 500 nm, e.g. in the range of 100nm to 200 nm. Such perforations may, for example, be formed by a focusedion beam (FIB) process step.

As already mentioned hereinabove, the graphene layer 104 may be providedfrom above or from below to cover the hole or holes (e.g. w.r.t. thesubstrate base as ‘bottom’ layer), for example as illustrated inrespectively FIG. 2 and FIG. 3 .

This hole or holes are covered by a graphene layer 104, e.g. a monolayerof graphene, or a multilayer (e.g. stack of monoatomic layers). Multipleholes may be covered by a continuous layer of graphene, or each hole maybe covered by a separate layer of graphene, e.g. a separate segment ofgraphene supported by the edges of the hole. A same hole may be coveredby graphene from different grains. It will be understood that coveringeach hole by a separate piece of graphene (mono)layer may avoid stressesover extended areas of the material, may be easier to manufacture, andmay improve the likelihood of obtaining a homogeneous, uniformly thick,unwrinkled and clean surface of graphene covering each hole separately.However, in so far that a piece of graphene layer can be obtained ofsufficient dimensions and sufficient quality, the manufacturing processmay be more efficient if multiple holes can be covered by a singlepiece. This concept is schematically illustrated by the two diagrams inFIG. 3 , in which, in the lower drawing, different segments of grapheneare used to cover two (depicted) holes respectively, while in the upperdrawing, a single extended, (substantially) continuous piece of graphenecovers the holes.

The graphene layer 104 forms a sample support onto which a sample ofinterest can be deposited for examination by charged particlemicroscopy, e.g. transmission electron microscopy. The heater element101 allows the sample to be heated, in some embodiments, in a preciselycontrollable manner (e.g. using a four-point probe method to monitor thetemperature during the heating), while being studied. The graphene has ahigh thermal conductivity, such that an efficient and uniform heating ofthe sample can be obtained, while undesired charge effects can beavoided thanks to its high electrical conductivity. On the other hand,the membrane 103 isolates the graphene, and thus also the sample, froman electrical current supplied via the heater and provides a robustsupport that can also withstand high temperature and/or ambientpressure.

For example, the device may comprise a conventional TEM sample holderwith a MEMS technology heater, as known in the art, that is specificallymodified to include the perforations covered by graphene. Compared to a(e.g.) copper TEM grid, such MEMS holders may typically comprise atleast one silicon nitride membrane (or similar alternative) to formelectron-transparent windows onto which the sample can be deposited foranalysis. Although such membrane may offer various aspects, such asproviding a structurally sound and reliable sample support (even underelevated pressure and/or temperature), it is not ideally suited forhigh-quality microscopy, since the typical thickness of such membranecan create a substantial background signal, while a poor uniformity ofthis layer, and thus also of the background signal, may lead to areduced signal quality and difficulties in performing quantitativemeasurements. By creating small holes in such membrane and coveringthese openings with a very thin graphene layer (e.g. a monoatomicgraphene layer), properties of the membrane can be combined with ahigh-quality electron transmission and heat dissipation achievable by agraphene sample support. Furthermore, as discussed in more detailhereinbelow, the present disclosure also provides, in a further aspect,a manufacturing process that can provide such graphene sample supportelements on the membrane with a very low thickness, e.g. less than (orequal to) 2 nm, a good flatness (e.g. wrinkling is prevented or reducedby the method), a good cleanness and/or a high uniformity.

The device may also comprise other features as known in the art for suchMEMS sample holders. For example, heat sink element(s) 117 may beincluded to improve the temperature stability and/or to obtain a moreuniform heating. Such heat sink element may be formed in or on themembrane, or in close proximity thereto. The device may comprise heatsensing element(s), e.g. to enable a measurement of the temperature in,on or near the observation region. As known in the art, such heatsensing element may comprise a thermo-sensitive resistor, thermocoupleor thermopile structure. Another example of an optional device featureis a pressure sensor, which may be formed by (e.g.) a metal thin filmresistor formed in or on the membrane, to detect a pressure of a gas atthe sample location. For example, such pressure sensing element maycomprise a metal thin film resistor that undergoes a change inelectrical resistance when deformed, such that an ambient pressure canbe determined from a measurement of its resistance (or impedance). Otherexamples include chemical sensing elements to detect specific compoundsand/or elements in the local environment where the sample is deposited.

The device may typically also comprise electrical contacts 114, e.g.contact pads, to connect the heater to an electrical power source,and/or to control and/or read sensor signals, which may be created byconventional semiconductor process techniques.

Referring to FIG. 5 , the device further comprises a cap 121, e.g.comprising a second substrate 126, to cover at least the membrane, suchthat a cavity 120 (a chamber) is formed between the cap and the membranewherein a sample can be isolated in a controllable gaseous environment.Additionally, it is to be noted that such, e.g. air-tight, chamber mayalso be suitable for use as a fluid cell without requiring substantialmodifications, since the physical requirements for containing a(possibly highly reactive and/or high pressure) gas are typically morestringent than those for containing a fluid environment.

Likewise, this cap may comprise a membrane 123, generally positionedover the same observation region(s), such that an incident beam used forimaging can pass through the sample. This membrane 123 may also beformed by a graphene (mono)layer (or stack of at least two such graphenelayers), or may comprise holes covered by a graphene layer(s) 124, e.g.in generally the same manner as discussed hereinabove. The device mayalso comprise an O-ring or other type of seal or gasket to fit betweenthe cap 121 and the primary device structure (the device part that isintegrated in and/or on the first substrate 110), such as to seal thesample chamber 120.

The device may furthermore comprise at least one channel 127, e.g.microfluidic channels, such that a fluid or gas can be introduced intothe chamber, and/or such that a flow of a gas or fluid of interestthrough the chamber can be provided (e.g. such that the sample can beexposed to the gas or fluid of interest), e.g. as schematically depictedin FIG. 5 . In other words, at least an inlet and an outlet channel maybe defined that connect to the chamber formed between the cap and themembrane. Such channels may be defined in the cap, in the primarysubstrate (and associated structures) in/on which the heater andmembrane are formed, or a combination of both. The term “cap” should notbe interpreted too narrowly, since the cap may (or may not) alsocomprise additional device features, e.g. a heater structure, a sensorelement, a heat sink, flow channels, flow regulators, etc.

Likewise, the device does not necessarily comprise a cap, but may beplaced in an encapsulating holder, forming a chamber around the device.Such holder may optionally also be provided with an inlet and an outletto allow a gas (or liquid) to flow through the chamber. This holder maycomprise transparent windows, e.g. window devices 130 (see e.g. FIG. 6), between which the device holding the sample may be placed to allow abeam to pass through the windows, the device, and the observation region102 thereof, to image a sample.

In a second aspect, the present disclosure relates to a method ofmanufacturing a sample support device for charged particle microscopy,e.g. transmission electron microscopy. For example, the method may be amethod of manufacturing a device in accordance with embodiments of thefirst aspect of the present disclosure.

FIG. 7 shows an illustrative method 10 in accordance with embodiments ofthe present disclosure.

The method 10 comprises providing 11 a substrate 110 having a heaterelement 101 integrated therein or thereon to heat, in use of the device,a sample when positioned in an observation region 102 of the device, andin which a membrane 103, e.g. a silicon nitride membrane, covers anopening in the heater element and/or substrate in an observation region102 thereof.

This step of providing lithe substrate and associated structures maycomprise obtaining a MEMS sample support device as presentlycommercially available, or the manufacturing of such device inaccordance with methods known in the art. For example, the heaterelement 101 may be formed from a metal layer deposited on a suitablesemiconductor substrate, e.g. a silicon wafer. The metal layer (or stackof layers) may be patterned to define the heater structure. The heaterelement may be encapsulated between two insulator layers 111, 112, orcovered by an insulator layer 111. In the observation region 102, theinsulator layer 111 may be thinned to form the membrane, or locallyremoved to subsequently deposit a separate layer to form the membrane.The substrate and metal layer may be patterned from the backside todefine a cavity underneath the membrane in the observation region, e.g.such that the membrane remains suspended over the cavity.

The method 1 comprises perforating 12 the membrane 103 to form at leastone (through)hole 105 therein. For example, this hole or holes may havea diameter in the range of 50 nm to 5 μm, in the range of 75 nm to 1 μm,e.g. in the range of 100 nm to 500 nm, e.g. in the range of 100 nm to300 nm. Perforating 12 the membrane may comprise creating the hole(s) bya focused ion beam (FIB), e.g. using FIB SEM. For example, a gallium(Ga) source may be used to generate the FIB beam, e.g. at 30 kV and 80pA. The hole or individual holes may (e.g.) be dimensioned according to(about) 10% of the average grain size of the graphene of the graphenelayer 104 to be transferred on the membrane (e.g. which may have a grainsize of about 2 μm). Grain sizes may vary, e.g. typically in the rangeof 2 to 4 μm, but not excluding less common sizes, e.g. even potentiallyup to 40 μm (without limitation thereto, e.g. not excluding largersizes, if available). An array of holes may be provided, e.g. a periodicarray, or a single hole in the membrane. For example, a low number oronly one hole may be in embodiments where a substantially sealed chamber120 is to be achieved (e.g. as discussed hereinabove), e.g. to preventleakage of gases and/or fluids from the chamber.

Optionally, the method may comprise a treatment of the unfinished device(i.e. before transferring 13 the graphene layer) with a plasma cleaning,e.g. using argon and/or oxygen plasma, to increase the hydrophilicity ofthe surface onto which the graphene layer is to be transferred. As anexample, a Fishione Model 1070 plasma cleaner may be used for an Ar/O₂plasma cleaning of 30 seconds (or, in another example, e.g. 3 minutes)at 30 sccm and 15 Watt (other settings, equipment and/or plasmatreatment methods not excluded).

Furthermore, the method may comprise protecting contacts (e.g. contactpads 114) of the unfinished device, e.g. using a (easily) removablecoating or protective tape. For example, Kapton tape or a similar tapefor sensitive materials (and that can withstand high temperatures) maybe used. This step may be carried out before the step of transferring 13the graphene layer, or specifically before a dry cleaning 31 stepthereof, discussed in detail hereinbelow, to protect the contacts duringa heat treatment in said step.

The method comprises transferring 13 a graphene layer 104 onto themembrane 103 to cover the at least one hole through the membrane to forma sample support onto which a sample of interest can be placed forstudy, e.g. for microscopy imaging.

Transferring 13 the graphene layer, e.g. a graphene monolayer, onto themembrane 103 may comprise a transfer method as illustrated in FIG. 8 .By this transfer method, a low graphene roughness and a good flatnesscan be achieved, e.g. avoiding or reducing wrinkles of the layer.

Transferring 13 the graphene layer may comprise obtaining 2 a metal foil32 onto which the graphene (mono)layer 104 is provided. The graphenelayer 104 may be a monolayer, e.g. having large single-crystallinedomains. The graphene layer 104 may be grown by chemical vapordeposition (CVD) onto the metal foil 32, e.g. as known in the art.

For example, the metal foil with graphene layer may be a monolayer CVDgraphene on copper (Cu), which is for example commercially availablefrom Graphenea Inc., Cambridge (USA). The same or similar product may beavailable from other high-quality graphene providers for research and/orhigh-tech applications. For example, such graphene foils may beavailable in a size of about 1 cm² or more, e.g. 15 cm by 15 cm, e.g. 10mm by 10 mm.

Obtaining 2 the metal foil with the graphene (mono)layer thereon maycomprise forming a graphene layer (e.g. monolayer) on a metal foil, e.g.using chemical vapor deposition. Many variations are known in the art tosynthesize a graphene layer onto a metal substrate, e.g. using CVD. Forexample, methane gas can be used as carbon source for such depositionprocess, and alternatives may include e.g. petroleum asphalt. Hydrogenmay be used in the process to promote carbon deposition (e.g. using acombination of methane and hydrogen at a suitably tuned flow ratio). Thehydrogen may corrode amorphous carbon and improve the quality of thedeposited graphene, but should be used in moderation to avoid corrosionof the graphene and/or damage to the formed crystal lattice. The carriergas may comprise an inert gas, e.g. argon, for example in combinationwith hydrogen. Optionally, a catalyst may be used, such as ironnanoparticles, nickel foam and/or gallium vapor. Not only the gas flowratio is typically tuned to optimize results, but also the ambientpressure, temperature and chamber material may need to be considered toachieve high-quality layers. Quartz may be a suitable material for theCVD chamber and auxiliary tubing. These deposition processes arewell-known in the art, and the skilled person is capable of determiningsuch parameters with straightforward experimentation and the knowledgereadily available in the field.

Transferring 13 the graphene layer may comprise mechanically flattening3 the metal foil 32 (supporting the graphene layer 104), e.g. bycompressing the foil (with graphene layer) between two flat surfaces 21.For example (without limitation thereto), the metal foil may be placedbetween two clean microscope slides, e.g. previously cleaned withethanol and acetone to avoid contamination of the graphene layer 104.

Transferring 13 the graphene may comprise stabilizing the graphene layer104 by applying 4 a layer 33 of a cellulose-based polymer, e.g.cellulose acetate butyrate (CAB), onto the graphene layer 104. Otherexamples of cellulose-based polymers may include ethyl cellulose (EC),cellulose acetate (CA) and cellulose acetate propionate (CAPr). Otherexamples may comprise, for example, formvar (polyvinyl formal), butvar(polyvinyl butyral resin) and/or glycol, without limitation ofembodiments to the mentioned examples.

The layer 33 may be a thin layer, e.g. having a thickness in the rangeof 10 nm to 500 μm, in the range of 15 nm to 1 μm, e.g. in the range of15 nm to 100 nm, e.g. about 20 nm. For most purposes, a layer of nm maybe considered sufficient, even though higher values are not necessarilydetrimental. However, the final step of cleaning away thecellulose-based polymer may be easier, faster and/or less prone toleaving contamination behind when a layer thickness in the lower rangesmentioned hereinabove is selected.

The step of applying the cellulose-based polymer layer onto the graphenelayer may comprise coating the metal foil having the graphene layerattached thereto with a solution of the cellulose-based polymer, e.g.using a dip-coating method.

For example, the metal foil with graphene may be dip-coated with acoating solution, e.g. a solution of g CAB (e.g. having an averagemolecular weight M_(n) of about 30000; e.g. as commercially availablefrom Merck KGgA; CAS no. 9004-36-8) in 100 mL ethyl acetate or anothersuitable solvent (e.g. a solution of 2.5 g/L CAB in ethyl acetate).Other suitable cellulose-based polymer solutions, e.g. in a suitablevolatile solvent, may be considered as well. Obviously, a homogenoussolution can be achieved by stirring or another type of agitation, e.g.using a magnetic stirrer. The solution of CAB may have a concentrationin the range of 1 g/L to 10 g/L, e.g. in the range of 1.5 g/L to 5 g/L,e.g. in the range of 2 g/L to 3 g/L, e.g. in the range of 2.3 g/L to 2.7g/L of CAB in ethyl acetate or another suitable solvent. The CAB mayhave a molecular weight (Mn) in the range of 12*10{circumflex over ( )}3to 70*10{circumflex over ( )}3, e.g. 12*10{circumflex over ( )}3, e.g.30*10{circumflex over ( )}3, e.g. 70*10{circumflex over ( )}3.

For example, in such dip-coating process to apply 4 the layer 33 of thecellulose-based polymer onto the graphene layer, the metal foil 32 withgraphene layer 104 may be immersed in the coating solution, e.g. at asubstantially constant (i.e. jitter-free) speed. After a predeterminedtime in the solution, the substrate is pulled out of the solution suchthat a thin layer of the cellulose-based polymer deposits on thesubstrate during the pull-up. Again this pulling up may be carried out,in some embodiments, at a constant (jitter-free) speed. This speed canbe tuned to determine the thickness of the coating, and/or the immersionprocess can be repeated (one or more times) to achieve a desired layerthickness. Excess liquid may then be drained from the surface, and thesolvent (e.g. ethyl acetate) can be allowed to evaporate, thus formingthe thin layer.

The layer of the cellulose-based polymer may be applied such as to reacha layer thickness (of the cellulose-based polymer) in the range of 10 nmto 100 nm, in the range of 15 nm to 30 nm, e.g. about 20 nm. In someembodiments, a dip-coating that the thickness of the layer can be easilyand reproducibly controlled in this range, e.g. to achieve a layerthickness of about 20 nm.

The cellulose-based polymer, e.g. CAB, on the side of the metal foil 32that is opposite of the side covered by the graphene layer 104 may thenbe removed from the metal foil, e.g. by a suitable solvent such asacetone. For example, dip-coating may produce a stack ofCAB-graphene-Cu-CAB, wherein the CAB layer directly on the metal foil isremoved to obtain substantially a stack of CAB-graphene-Cu. For example,the CAB (on the side where it is not desirable) may be removed using adrop of acetone, which is applied carefully to the side to be cleaned toavoid spreading to the topside of the CAB-graphene-Cu stack. A drop ofacetone may be deposited on a clean surface, e.g. a glass slide, and thebackside of the stack may be brought carefully into contact with theacetone with due care to avoid dispersion to the other side.

Thus, the graphene layer to be stabilized and protected is covered by acellulose-based polymer layer, while the other side of the metal foil issubstantially cleaned of this polymer, such that it can be exposed to anetchant.

The step of transferring the graphene may further comprise placing 5 themetal foil 32, having respectively the graphene layer 104 (directly) andthe cellulose-based polymer layer 33 (indirectly) provided on topthereof, in or on an etching solution 25 to dissolve the metal 32supporting the graphene layer 104. For example, the metal foil 32 may beplaced downside onto the surface of the etching solution to furtheravoid or minimize damage to the sensitive graphene layer, e.g. allowingthe foil to float in or on the etching solution.

The etching solution 25 may comprise ammonium persulfate ((NH₄)₂S₂O₈) ormay consist of a solution of ammonium persulfate, e.g. in (substantiallypure) water. It is an aspect that, thanks to the stabilization by thepolymer, a relatively high concentration, e.g. 3.4%, of ammoniumpersulfate can be used, which thus contributes to a speedy process. Thisalso has the aspect that the graphene is protected from damage and/orwrinkling.

However, the graphene transfer process is not necessarily limited to theexample discussed hereinabove (and further below). For example, a wettransfer process as known in the art may be used, e.g. using a (generic)polymer as intermediate carrier layer for the graphene and/or assacrificial layer. Such (generic) polymer may be etched away, e.g. insubstantially the same manner as discussed for cellulose-based polymerhereinabove.

A polymer-free transfer method as known in the art, e.g. which does notrequire a polymer to be added to the foil, may also be used. However,when a polymer-free transfer process is used, e.g. instead of thecellulose-based polymer transfer method described hereinabove, loweretchant concentrations may need to be used, e.g. about 10 times lower,e.g. in the range of 0.3 to 0.8%, e.g. about 0.34%, of ammoniumpersulfate, such that the cellulose-based polymer approach discussedhereinabove might be desirable (e.g. due to protection from damageand/or wrinkling, as discussed hereinabove). The necessitated use of alower concentration of the etching solvent may also have thedisadvantage that the corresponding time that is needed to etch away themetal foil may increase, e.g. about 12 hours or longer for aconcentration in the range of 0.3 to 0.8%. Nonetheless, it is an aspectof a polymer-free transfer process that no contamination and/or residueis created by a polymer that might otherwise be used in the process.Therefore, additional steps and/or specific care to remove suchcontamination and/or residue is not necessary as such in a polymer-freetransfer approach.

For example, the dissolution of the metal foil can be carried out inless than 4 hours, e.g. less than 2 hours, e.g. about an hour or evenless. Even though the etching time may, generally, depend on theconcentration of the etching solution, the stabilizing effect of thecellulose-based polymer enables the use of a relatively highconcentration, and thus a rapid etching.

For example, the etching solution 25, e.g. an ammonium persulfatesolution, may be heated to a temperature in the range of 30° to 50° C.,e.g. in the range of 35° C. to 45° C., e.g. about 40° C., e.g. tofurther increase the efficiency of the chemical etching process. Forexample, the solution may be prepared on a hotplate (e.g. at 40° C.)while being continuously stirred, e.g. using a magnetic stirrer (e.g. at650 rpm). However, it is to be noted that heating the etching solutionis not necessarily required, e.g. the same etching can be achieved atroom temperature, even though a faster etching can be achieved atelevated temperature.

The etching solution may be a solution of ammonium persulfate of 3.42 gin 100 mL (e.g. 34.2 g/L) in a suitable solvent, such as water, or suchas ultrapure water (UPW), e.g. according to the standards defined inASTM D5127 and/or SEMI F63. The concentration of the ammonium persulfatemay be in the range of g to 5 g per 100 mL solvent, e.g. in the range of0.3 g to 4 g per 100 mL solvent, e.g. in the range of 2 g to 4 g per 100mL solvent, e.g. in the range of 3.0 g to 3.5 g per 100 mL solvent, e.g.in the range of 3.3 g to 3.4 g per 100 mL solvent. While excessiveetching concentrations could potentially damage the graphene, it isnoted that higher concentrations may fasten the etching processconsiderably. However, if the exposure time is adjusted accordingly, acopper foil can be etched away with concentrations of etchant((NH₄)₂S₂O₈) as low as, e.g., 0.34 g/100 mL UPW.

In order to prevent contamination by the dissolved metal of the metalfoil, the graphene transfer process may comprise diluting and/orneutralizing 6 the etching solution 25 before removing the graphenelayer, which is now only attached to (and stabilized by) the remainingcellulose-based polymer layer 33. For example, this dilution maycomprise a gradual dilution (and/or neutralization) using water, e.g.ultrapure water, to remove the metal, e.g. copper, residues. Forexample, clean solvent, e.g. ultrapure water, may be added repeatedly,e.g. while siphoning off excess solution (e.g. using a pipette).

The etching solvent may thus be removed by exchanging it, e.g. in steps,with water (such as UPW), for example until the pH has reached a valuesufficiently close to pure water, e.g. in the range of 6.9 to 7.1 orsubstantially 7.0. For example, the etchant may be exchanged (e.g.repeatedly and/or stepwise) with ultrapure water, until a pH in therange of 5.8 to 6.1, e.g. 5.8 to 5.9, e.g. about 5.85, is reached.

For example, the etching solvent may be exchanged with water repeatedlyuntil the pH value is increased from an acid environment (e.g. about 3)to neutral (e.g. about 7).

This step avoids damage to the target substrate, i.e. the membrane ontowhich the graphene is transferred, and may prevent or reducecontamination by residues of the dissolved metal.

While the etching solvent may be alternatively exchanged with anothersolvent, such as isopropanol, ethanol and/or acetone, experiments haveshown that these alternatives could potentially lead to a highercontamination and/or could pose problems, e.g. of the graphene tendingto attach more easily to the wall of the recipient. Nonetheless,embodiments using another solvent than water for dilution and/orexchange of the etching solution are not necessarily excluded.

The graphene transfer step may further comprise depositing 7 thegraphene layer 104 (directly) onto the membrane 103 to cover the atleast one hole 105 through the membrane. It is an aspect of a method inaccordance with embodiments that the graphene layer, floating in or onthe diluted etching solution, can be easily scooped onto the targetsubstrate or structure.

Referring to FIG. 9 , depositing 7 the graphene layer onto the targetmembrane may comprise placing 27 the target substrate with theperforated membrane underneath the graphene layer floating in thesolution e.g. the diluted and/or neutralized etching solution 25 (e.g.substantially exchanged by the solvent, such as water, or such as UPW),and removing 28 the solution until the graphene layer settles over theperforation hole and (along its edges) onto the membrane 103. Forexample, the solution may be removed by a (e.g. microliter) pipette, oranother suitable method, such as by a pump or by evaporation (or anycombination thereof).

The target substrate with the perforated membrane may, for example, beplaced 27 on top of a piece of filter paper 26 or other suitableprotective (e.g. shock absorbing) material at the bottom of therecipient containing the solution 25, and the graphene layer 104 canthus be softly lowered on top of the substrate or structure by gentlyremoving the solution without risk (or with reduced risk) of damage. Itwill also be appreciated that such filter paper 26 or similar carriermaterial can be used to easily manipulate the substrate or structure,e.g. to remove the construct from the recipient once the graphene layerhas been deposited thereon. Furthermore, the filter paper 26 may beprovided with a film, e.g. a parafilm, to attach the target substrateonto the filter paper (i.e. preventing the target substrate fromfloating in the solution). Alternatively, such film may be applieddirectly to the (bottom of) the recipient to temporarily attach thetarget substrate while the graphene is deposited onto the targetlocation on the membrane.

Even though reference is made to the ‘solution’ for the sake of clarity,it will be understood that the ‘solution’ at this stage in the processmay be substantially nothing more than water, e.g. if the aforementionedstep of removing 6 (by diluting and/or neutralizing) the etching solventwas carried out effectively.

It will also be understood that the position of the graphene layer,while lowering it on top of the substrate or structure can be controlledduring this step, e.g. by gentle nudges and/or by controlling the(direction of the) flow by which the solution is being removed, suchthat a good alignment of the graphene layer on the target (hole in themembrane) can be obtained.

The graphene transfer step may further comprise drying 9 the targetsubstrate with the graphene layer 104 deposited over the hole(s) in themembrane. For example, by placing the construct on a hotplate, e.g. at atemperature in the range of 25° C. to 50° C., e.g. in the range of 30°C. to 40° C., e.g. about 35° C. For example, about one hour may sufficeto dry the device (without limitation thereto). It is to be noted that,when placed on a filter paper or other suitable carrier material, it isnot only easy to remove the construct from the recipient that containedthe diluted etching solution (e.g. substantially water) for drying, butthat the construct may also be protected from direct heat in the dryingprocess. It is also noted that the drying may also be carried out insitu, e.g. without removing from the recipient, to avoid any damage dueto manipulation.

The transfer step may further comprise a dry cleaning 31 (a dry heatingtreatment) to remove the cellulose-based polymer layer 33.

In this step, the construct (i.e. the substrate with the graphene layer104 placed on the membrane and the remaining cellulose-based polymer 33on the graphene) may be brought into direct physical contact with, e.g.may be embedded in, activated carbon 310. For example, the activatedcarbon may be provided in a recipient (e.g. a petri dish) or pile, a pitmay be formed in the activated carbon, and the construct may be buriedin this pit, substantially covering the construct from all sides by theactivated carbon (i.e. closing the pit). For example, a heap of 0.7 cmactivated carbon may be provided, and the construct may be buried at adepth of about 0.5 cm in this heap. Alternatively, the activated carbonmay be provided in the form of a film, a tape, a strip, a sheet, orother similar (e.g. substantially planar) form, which is brought intodirect contact with (at least) the graphene layer and/or the polymer(e.g. CAB) thereon. The activated carbon film (or other substantiallyplanar form) is not necessarily exclusively composed of activatedcarbon, e.g. may comprise a polymer or other binding medium, such that asufficiently stable film (or other planar form) is obtained that canwithstand the elevated temperature discussed hereinbelow. For example, apolymer film that can (at least for some time, e.g. at least minutes,such as at least half an hour) withstand a temperature of e.g. 300° C.and that contains activated carbon in its matrix may be used.

The activated carbon 310 in which the construct is embedded may then beheated, e.g. to a temperature in the range of 200° C. to 350° C., e.g.in the range of 250° C. to 300° C., for example at a rate of 5° C. perminute. Specifically, the target temperature is higher than the meltingtemperature of the cellulose-based polymer, but conservatively selectedto avoid damage to the graphene and/or the target substrate or structure(e.g. the membrane). For example, the target temperature may be at least5° C., such as at least above (an upper limit of) the meltingtemperature. This heat treatment may be carried out in a vacuum chamber.

This target temperature is maintained for a sufficient time, e.g. atleast 30 minutes, e.g. at least 1 hour, e.g. at least 2 hours, 4 hours,e.g. at least 8 hours, e.g. for a time in the range of 10 hours to 24hours, e.g. hours. After cooling down (and removing of the constructfrom the carbon, or vice versa), the sample support device remains, withthe graphene layer positioned over the perforated membrane, while thepolymer is cleaned away.

By heating in activated carbon, e.g. for 15 h at 300° C., thecellulose-based polymer, e.g. CAB, and potentially other contaminantscan be efficiently removed. The melting temperature of CAB is between170° C. and 240° C., such that a temperature slightly above the meltingtemperature of the polymer can liquefy the material and allow it to beabsorbed by the carbon. It is to be noted that other cellulose-basedpolymers may equally be suitable for a method in accordance withembodiments, and the temperature of the thermal treatment can beadjusted accordingly.

Alternatively, (or additionally) the step of dry cleaning 31 to removethe cellulose-based polymer layer 33 and possible contaminants maycomprise an annealing treatment in vacuum (e.g. a high vacuum).

The method may also comprise a rapid cooling of the activated carbonwith the device therein embedded (or, more generally, with the activatedcarbon in direct contact with the device), after this heat treatmentstep, e.g. by exposure to liquid nitrogen, e.g. to avoid degassing.

The contacts of the device may be protected during this step by aprotective coating or tape, as discussed hereinabove. Such protectivetape or coating may subsequently be removed.

The quality of gas flow cells in accordance with embodiments of thepresent disclosure were experimentally tested in the illustrativeexperiments discussed hereinbelow. Devices were manufactured inaccordance with the procedures discussed hereinabove, see e.g. FIG. 7 .Deposition of one as well as two layers of graphene, forming acontinuous seal, was successfully achieved in a single chip for use astop or bottom component of a gas flow cell, as well as in both a top andbottom component of a cell, e.g. as illustrated in FIG. 5 . The gas flowcells thus obtained were successfully used in gas experiments.

It is to be noted that the coverage of the holes by the graphene layersis particularly important. It was found that by depositing two layers ofgraphene (e.g. successively, e.g. on top of each other), the likelihoodof obtaining a functional and, in use, stable device could be increasedsignificantly. The presence of graphene in the observation window can beconfirmed by observing its characteristic diffraction pattern (i.e.typical graphene diffraction spots).

Particularly, the effect of the expected diffraction pattern of a doublegraphene layer can be observed in highly defocused transmission electronmicroscopy (TEM) images. FIG. 10 shows a conventional bright-fieldtransmission electron microscopy (BF-TEM) image of a hole (withdimensions in the order of 0.5 to 2 μm, approximately) covered by twolayers of graphene, in a proof-of-concept gas-cell in accordance withembodiments of the present disclosure. In this example, it can be notedthat the shape and dimensions of the hole(s) is/are not necessarilysymmetric and/or isotropic. For example, drift of the FIB instrumentused to drill the hole(s) may cause an elongated shape of the hole(s),as can be seen in FIG. 10 . Therefore, the freestanding and/orunsupported graphene layer sections that extend over and cover thehole(s) (in other words, the sample supports) are not necessarilysymmetric (under mirroring, point reflection, rotation and/or othersymmetries) either.

A corresponding highly defocused BF-TEM image is shown in FIG. 11 , inwhich diffraction spots can be seen that are in line with theexpectations for a double layer of graphene, thus confirming thepresence of the two layers of graphene as sample support.

Different perforation sizes, created by a focused ion beam (FIB) method,were evaluated, which confirmed that functional devices could beconstructed for perforation sizes in the range of at least 20 nm to 1μm. In these experimental tests, a maximum pressure (at pumping station)was applied of 3.4×10⁻⁶ hPa, to reach a pressure in the TEM system of1.18×10⁻⁵ Pa, i.e. the minimum achievable pressure of the microscopesystem. Corresponding to the electron microscope's specifications, aworking range of 1.18×10⁻⁵ Pa to 4.9×10⁻⁵ Pa was maintained. The maximumpressure applied inside the gas cell was 101325 Pa (i.e. correspondingto atmospheric pressure). The following table illustrates the pressureinside the TEM chamber as function of temperature, in tests of a gasflow cell device in accordance with embodiments with a double graphenelayer as discussed hereinabove:

Temperature Pressure in the TEM Room temperature 1.18 × 10⁻⁵ Pa  50° C.1.18 × 10⁻⁵ Pa 100° C. 1.18 × 10⁻⁵ Pa 200° C. 1.18 × 10⁻⁵ Pa 300° C.1.18 × 10⁻⁵ Pa 400° C. 1.18 × 10⁻⁵ Pa 500° C. 1.18 × 10⁻⁵ Pa 600° C.1.18 × 10⁻⁵ Pa 700° C. 1.64 × 10⁻⁵ Pa to 2.42 × 10⁻⁵ Pa (Increment over10 minutes; attributable to degradation)

This illustrates that a device in accordance with embodiments isparticularly suitable for maintaining and withstanding a high pressureinside the cell relative to the low pressure external TEM environment,at substantially high temperatures, e.g. 700° C. It will be appreciatedthat a temperature of 700° C. is greater than the temperature at whichtypical (and even most) gas-cell experiments are performed. FIG. 12shows three TEM images of a graphene support after reaching the finaltemperature of 700° C. Even though the pressurized containment fails atthis point (e.g. over about 10 minutes at this temperature; at least inthe present experiments), high-temperature degradation of the graphenesupport is not immediately apparent and/or extensive. Particularly, theimages shown in FIG. 12 were obtained after the pressure/temperaturetesting procedure discussed hereinabove, and correspond to threedifferent regions of the same chip. However, it can be observed that thegraphene layer has substantially survived the strong pressure differenceand high temperature conditions imposed by this test.

1. A sample support device for charged particle microscopy, the devicecomprising: a substrate, a heater element and/or biasing electrodeintegrated in or on the substrate to heat and/or bias a sample ofinterest when positioned in an observation region of the device, amembrane covering an opening in the heater element and/or substrate inthe observation region of the device, wherein said membrane isperforated to form at least one hole therein, a graphene layer coveringsaid at least one hole in said membrane to form a sample support toplace the sample thereon for study, and a cap to cover at least themembrane such that a chamber is formed between the cap and the membranewherein a sample can be isolated in a controllable gaseous environment,wherein said at least one hole in said membrane is at least five timessmaller in area than said opening, covered by said membrane, forming theobservation region.
 2. The sample support device of claim 1, whereinsaid graphene layer has a thickness of less than 2 nm.
 3. The samplesupport device of claim 1, wherein said membrane has a thickness in therange of 2 nm to 1 μm.
 4. The sample support device of claim 1, whereinsaid graphene layer comprises a number n of stacked graphene layers, inwhich n is in the range of 2 to
 5. 5. The sample support device of claim1, wherein said membrane comprises an amorphous silicon nitride layer.6. The sample support device of claim 1, wherein said at least one holein said membrane is at least ten times smaller in diameter than saidopening, covered by said membrane, forming the observation region,and/or wherein said at least one hole is at least ten times smaller inarea than said opening, and/or wherein said at least one hole is atleast five times smaller in diameter than said opening, and/or whereinsaid at least one hole has a diameter in the range of 50 nm to 5 μm. 7.The sample support device of claim 1, wherein said heater elementcomprises a spiral-shaped and/or meandering electrical conductor.
 8. Thesample support device of claim 1, comprising at least one heat sinkelement to improve the temperature stability and/or heating uniformityof a sample when placed on the graphene layer and heated by the heaterelement.
 9. The sample support device of claim 8, comprising at leastone channel to provide a flow of a gas or a fluid of interest throughsaid chamber.
 10. A method of manufacturing a sample support device forcharged particle microscopy, the method comprising: providing asubstrate having a heater element and/or biasing electrode integratedtherein or thereon to heat and/or bias a sample when positioned in anobservation region of the device, and comprising a membrane covering anopening in the heater element and/or the biasing electrode and/or thesubstrate in said observation region, perforating said membrane to format least one hole through the membrane using a focused ion beam processstep, transferring a graphene layer onto said membrane to cover said atleast one hole in said membrane such that the graphene layer forms asample support onto which a sample of interest can be placed for study,and covering at least the membrane by a cap such that a chamber isformed between the cap and the membrane wherein the sample can beisolated in a controllable gaseous environment, wherein said at leastone hole in said membrane is at least five times smaller in area thansaid opening, covered by said membrane, forming the observation region.11. The method of claim 10, wherein transferring the graphene layer ontothe membrane comprises a polymer-free transfer process.
 12. The methodof claim 10, wherein transferring the graphene layer onto the membranecomprises: obtaining a metal foil onto which the graphene layer isprovided, stabilizing the graphene layer by applying a layer of acellulose-based polymer onto the graphene layer, placing the metal foilhaving respectively the graphene layer and the cellulose-based polymerlayer stacked thereon in or on an etching solution to dissolve the metalfoil supporting the graphene layer, diluting and/or neutralizing theetching solution after the metal foil has been dissolved, depositing thegraphene layer directly onto the membrane by placing the substrateunderneath the graphene layer floating in or on the diluted and/orneutralized etching solution and removing the diluted and/or neutralizedetching solution until the graphene layer settles onto the membrane tocover said at least one hole.
 13. The method of claim 10, whereintransferring the graphene layer onto the membrane comprises dry cleaningthe substrate with the graphene layer deposited on the membrane toremove the cellulose-based polymer layer, referred to in claim 12, oranother polymer layer used as temporary carrier of the graphene layer insaid transferring, wherein said dry cleaning comprises: bringing thesubstrate with the graphene layer on the membrane into direct physicalcontact with activated carbon and heating the substrate with theactivated carbon in contact therewith, and/or applying an annealingtreatment in vacuum.
 14. The method of claim 13, wherein the activatedcarbon and the embedded substrate are heated to a temperature at least5° C. higher than the melting temperature of the cellulose-based polymerand said temperature is maintained for at least 30 minutes and/or saidtemperature is maintained for at least 4 hours.
 15. The method of claim13, comprising drying the substrate with the graphene layer deposited onthe membrane before said step of dry cleaning.
 16. The method of claim12, wherein placing said metal foil having respectively the graphenelayer and the cellulose-based polymer layer stacked thereon in or on theetching solution comprises placing the metal foil in or on the etchingsolution with the metal foil directed downward, such that the graphenelayer can settle onto the membrane without inversion in the step ofdepositing the graphene layer.
 17. The method of claim 12, whereinapplying said cellulose-based polymer layer comprises coating the metalfoil having the graphene layer attached thereto with a solution of thecellulose-based polymer using a dip-coating or spin-coating method, and,when using said dip-coating method, removing the cellulose-based polymerthat was dip-coated directly onto the metal foil on the side opposite ofthe side where the graphene layer is provided such that the metal foilis exposed and the graphene layer remains covered by the cellulose-basedpolymer.